The invention relates to pharmaceutically effective hydroxylamine derivatives, which are useful in the treatment of vascular diseases.
The invention relates to the use of compounds of general formulae (I), (II) and (III).
    R1 and R2 independently represent a hydrogen atom or a straight or branched C1-6 alkyl group optionally substituted with a phenyl group, or R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatoms, which heterocyclic ring is optionally substituted with one or more hydroxy, oxo or benzyl groups,    A represents a phenyl group optionally substituted with one or more C1-4 alkyl, C1-4 haloalkyl or nitro groups or halogen atoms, or a 5-6 membered heteroaromatic ring containing one or more nitrogen, oxygen or sulfur heteroatoms, optionally having N-oxide structure on the nitrogen heteroatom,    n is zero, 1 or 2,    z is zero or 1,    in compounds of general formulae (I), X represents a halogen atom or —NR4R5 group, where R4 and R5 independently represent a hydrogen atom or a straight or branched C1-6 alkyl group,    in compounds of general formulae (II), X refers to oxygen atom,    R3 represents a hydrogen atom or a straight or branched C1-6 alkyl group, Y represents a hydrogen atom or hydroxy group, halogen atom or C1-22 acyloxy group, with the restriction that if R4 and R5 are simultaneously hydrogen atoms, Y is other than hydroxy group,    with the proviso that in compounds of general formulae (I) and (II) where Y is other than halogen,
a) R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered, saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatom, which heterocyclic ring is substituted with one or more hydroxy, oxo or benzyl groups and/or
b) A is a N-containing heteroaromatic ring, which has N-oxide structure on the nitrogen heteroatom, and/or
c) z is 1,    with the further proviso that if X is halo and Y is hydroxy or acyloxy in compounds of general formulae (I),
R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered, saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatom, which heterocyclic ring is substituted with one or more hydroxy, oxo or benzyl groups and    with the proviso for compounds of general formulae (III) that    if R1 and R2 independently represent a hydrogen atom or a straight or branched C1-6 alkyl group optionally substituted with a phenyl group, or together with the nitrogen atom attached thereto form a 5-7 membered saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatoms, then A is a heteroaromatic ring containing oxygen or sulfur heteroatom or an N-containing heteroaromatic ring having N-oxide structure on the nitrogen heteroatom and    if A is a phenyl group optionally substituted with one or more C1-4 alkyl, C1-4 haloalkyl or nitro groups or halogen atoms, or a 5-6 membered N-containing heteroaromatic ring, then R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered, saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatom, which heterocyclic ring is substituted with one or more hydroxy, oxo or benzyl groups,    and of the salts and optically active forms of the above compounds for the production of pharmaceutical products used in the treatment and/or prevention of vascular diseases or diseases related to vascular disorders.
Compounds of similar structure are known from WO 97/16439. These compounds increase molecular chaperon expression, or molecular chaperon activity in cell exposed to a physiological stress. Due to this characteristic, they are useful for the treatment of diseases connected with the functioning of the chaperon system.
The protective and regenerating effect that compounds of similar structures have for vascular endothelial cells is known from WO 98/06400. These compounds are primarily useful for the prevention of damage caused by ischemia and for the treatment of cardiovascular and cerebrovascular diseases.
We have found that when the hydroxylamine derivatives described in the cited literature are chemically modified, preferably in such a way that, according to general formulae (I), (II) and (III) above,
1) a halogen atom is introduced into the propylene group of the aminopropyl group connected to the hydroxylamine part as substituent and/or
2) N-oxide is formed on the nitrogen atoms in the terminal groups of the molecule, namely on the nitrogen atom connected to the propylene group of the above mentioned aminopropyl group and/or on the nitrogen atom located in the heteroaromatic ring of the molecule,    then the resulting products are hydroxylamine derivatives which possess much more favorable pharmacological properties against vascular illnesses than known compounds which have been found to be useful for this purpose. Namely, the effect of these compounds is more intensive than that of the known prior art compounds used for similar purposes. Therefore they are especially useful as active ingredients in the treatment or prevention of vascular diseases or diseases associated with vascular disorders.
Based on this observation, this invention relates to the use of compounds of general formulae (I), (II) and (III)—where R1R2R3, A, X, Y, n and z are as above—, and to the use of the salts and optically active forms of the above compounds for the production of pharmaceutical products for the treatment and/or prevention of vascular diseases or diseases associated with vascular disorders.
A considerable part of compounds of general formulae (I), (II) and (III) are novel compounds.
Novel compounds are compounds of general formulae (I) wherein R1 and R2 independently represent a hydrogen atom or a straight or branched C1-6 alkyl group optionally substituted with a phenyl group, or R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatoms, which heterocyclic ring is optionally substituted with one or more hydroxy, oxo or benzyl groups,    A represents a phenyl group optionally substituted with one or more C1-4 alkyl, C1-4 haloalkyl or nitro groups or halogen atoms, or a 5-6 membered heteroaromatic ring containing one or more nitrogen, oxygen or sulfur heteroatoms, optionally having N-oxide structure on the nitrogen heteroatom,    n is zero, 1 or 2,    z is zero or 1,    X represents a halogen atom or —NR4R5 group, where R4 and R5 independently represent a hydrogen atom or a straight or branched C1-6 alkyl group,    Y represents a hydrogen atom or hydroxy group, halogen atom or C1-22 acyloxy group, with the restriction that if R4 and R5 are simultaneously hydrogen atoms, then Y is other than hydroxy group,    with the proviso that
a) if Y is hydrogen and/or X is a —NR4R5 group, where R4 and R5 have the above meanings,
R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered, saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatom, which heterocyclic ring is substituted with one or more hydroxy, oxo or benzyl groups and/or
A is a N-containing heteroaromatic ring, which has N-oxide structure on the nitrogen heteroatom, or
b) if X is halo and Y is hydroxy or acyloxy,
R1 and R2together with the nitrogen atom attached thereto form a 5-7 membered, saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatom, which heterocyclic ring is substituted with one or more hydroxy, oxo or benzyl groups,    and the stereoisomers of the above compounds and their salts.
Novel compounds are compounds of general formulae (II) wherein R1 and R2 independently represent a hydrogen atom or a straight or branched C1-6 alkyl group optionally substituted with a phenyl group, or R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatoms, which heterocyclic ring is optionally substituted with one or more hydroxy, oxo or benzyl groups,    A represents a phenyl group optionally substituted with one or more C1-4 alkyl, C1-4 haloalkyl or nitro groups or halogen atoms, or a 5-6 membered heteroaromatic ring containing one or more nitrogen, oxygen or sulfur heteroatoms, optionally having N-oxide structure on the nitrogen heteroatom,    n is zero, 1 or 2,    z is zero or 1,    X represents an oxygen atom,    R3 represents a hydrogen atom or a straight or branched C1-6 alkyl group,    Y represents a hydrogen atom or hydroxy group, halogen atom or C1-22 acyloxy group, with the proviso that if Y is other than halo,
R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered, saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatom, which heterocyclic ring is substituted with one or more hydroxy, oxo or benzyl groups and/or
A is a N-containing heteroaromatic ring, which has N-oxide structure on the nitrogen heteroatom,    and the stereoisomers of the above compounds and their salts.
Novel compounds are compounds of general formulae (III) wherein R1 and R2 independently represent a hydrogen atom or a straight or branched C1-6 alkyl group optionally substituted with a phenyl group, or R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatoms, which heterocyclic ring is optionally substituted with one or more hydroxy, oxo or benzyl groups,    A represents a phenyl group optionally substituted with one or more C1-4 alkyl, C1-4 haloalkyl or nitro groups or halogen atoms, or a 5-6 membered heteroaromatic ring containing one or more nitrogen, oxygen or sulfur heteroatoms, optionally having N-oxide structure on the nitrogen heteroatom,    n is zero, 1 or 2,    z is zero or 1,    with the proviso that    if R1 and R2 independently represent a hydrogen atom or a straight or branched C1-6 alkyl group optionally substituted with a phenyl group,    or together with the nitrogen atom attached thereto form a 5-7 membered saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatoms, then 5 A is a heteroaromatic ring containing oxygen or sulfur heteroatom or an N-containing heteroaromatic ring having N-oxide structure on the nitrogen heteroatom and if A is a phenyl group optionally substituted with one or more C1-4 alkyl, C1-4 haloalkyl or nitro groups or halogen atoms, or a 5-6 membered N-containing heteroaromatic ring, then R1 and R2 together with the nitrogen atom attached thereto form a 5-7 membered, saturated heterocyclic ring optionally containing further nitrogen and/or oxygen heteroatom, which heterocyclic ring is substituted with one or more hydroxy, oxo or benzyl groups,and the stereoisomers of the above compounds and their salts.
The invention relates to the above compounds. The invention further relates to 15 pharmaceutical products that contain as active ingredient compounds of general formulae (I), (II) and (III), or their stereoisomers, or their salts, where R1, R2, R3, A, X, Y, n and z are as defined above.
The following compounds of the Invention are especially preferable:    1. N-[3-(1-piperidinyl)propoxy]-pyridin-1-oxide-3-carboxamidine    2. N-[3-(1-piperidinyl)propoxy]-pyridin-1-oxide-3-carboximidoyl chloride    3. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-N′-n-butyl-pyridin-1-oxide-4-carboxamidine    4. N-[3-(1-oxido-1-piperidinyl)propoxy]-3-nitro-benzimidoyl-chloride dihydrate    5. 2-chloro-N-[3-(4-oxido-4-morpholinyl)propoxy]-benzimidoyl chloride    6. (R,S)-5,6-dihydro-5-[(1-piperidinyl)methyl]-3-(1-oxido-3-pyridyl)-4H-1,2,4-oxadiazine    7. 5,6-dihydro-5-[(4-benzyl-1-piperidinyl)methyl]-3-(3-pyridyl)-4H-1,2,4-oxadiazine    8. (R) or (S)-5,6-dihydro-5-[(2-oxo-1-piperidinyl)methyl]-3-(3-pyridyl)-4H-1,2,4-oxadiazine    9. (+)-5,6-dihydro-5-[(1-piperidlnyl)methyl]-3-(1-oxido-3-pyridyl)4H-1,2,4-oxadiazine    10. (R) or (S)-5,6-dihydro-5-[(1-oxido-1-piperidinyl)methyl]-3-(1-oxido-3-pyridyl)-4H-1,2,4-oxadiazine    11. 5.6-dihydro-5-[(4-hydroxy-1-piperidinyl)methyl]-3-(3-pyridyl)-4H-1,2,4-oxadiazine    12. N-[2-chloro-3-(1-piperidinly)propoxyl]-benzimidoyl chloride hydrochloride    13. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-pyridin-1-oxide-3-carboxamide            The biological effects of the compounds of the invention were tested by the following experiments:        Wounding migration assay in endothelial cell culture        
The effect of the compounds of the invention on the wounded monolayers of human umbilical vein endothelial cells (HUVEC) were studied in a cell culture system (in vitro). After reaching confluence, the HUVEC cells were wounded according to the method of Yamamura et al (J. Surgical Res. 63, 349-354, 1996). The number of migrated cells were registered using computerized image analysis 24 hours after wounding in the absence and presence of the active agents under testing in a concentration of 10−6 M. The active ingredient described in publication no. WO 98/06400, namely 5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine was used as reference compound. The obtained results are given in Table 1.
TABLE 1cell/mm2No. of the compound24 hoursReference30445848952115112601354In the following, we give the results of the test of blood vessel relaxing effect, performed in vitro on rat vessels, and also the morpholocial results of the thoracic aorta.
Three-month-old, genetically hypertonic (SH) Wistar Okamoto rats were treated for one month with various test compounds. Thereafter the functional and morphological tests were performed.
The Vaso-relaxing Effect of the Compounds of the Invention on the Thoracic Aorta of SH Rats (in Vitro Testing)
The test was performed by the method known from the literature [Japan J. Pharmacol., 59, 339-347 (1992)]. The SH rats were anesthetized with Nembutal (40 mg/kg, i. p.), then the thoracic aorta was removed and placed in oxygenized (95% O2+5% CO2) Krebs-Henseleit solution. The composition of the solution (mM): NaCl 118, KCl 4,7, CaCl2 2,52, MgSO4 1,64, NaHCO3 24,88, KH2PO4 1,18, glucose 5,5. The 3-mm-long aorta rings were suspended in a 20 ml organ bath of 37° C. The resting tension was 1 g, which was maintained throughout the equilibration. During the 1 hour equilibration, the medium was changed in every 20 minutes. The vessels were contracted with 10−6 M methoxamine (approx. 80% of maximal contraction). After reaching the maximal contraction, we tested the vasodilation resulting as the effect of the acetylcholine (Ach) (10−6-10−4 M), which informed us about the condition of the endothelium of the vessel wall. The contraction force was measured by an isometric strain gauge (SG-01D, Experimentia Ltd), and was registered on an OH-850 polygraph (Radelkis). At this time again, 5,6-dihydro-5-(1-piperidinyi)-methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine as described in WO 98/06400 was used as a reference compound. The results of these tests are summarized in Table 2.
TABLE 2The vessel relaxing effect of the compounds of the invention on thethoracic aorta of SH rats (in vitro testing)MaterialsAch doses (M)Doses10-610-510-4SH control55.157.272.0n = 10Reference77.480.281.7n = 12:20 mg/kgCompound no. 4.82.584.988.1n = 11; 5 mg/kgCompound no. 8.80.388.089.2n = 11; 20 mg/kgCompound no. 9.87.087.993.2n = 10; 5 mg/kgCompound no. 11.79.785.186.0n = 12; 10 mg/kgCompound no. 12.82.383.580.4n = 12; 20 mg/kgCompound no. 13.88.490.395.2n = 10
As the table shows, we registered a 30% relaxation decrease in the case of untreated hypertonic animals, which is the result of hypertonic-induced endothelial damage. The test compounds improved the relaxation properties of the vessels significantly, which is the result of the improved functioning of the endothelium, due to the relative increase of the endothelium-related relaxation factors.
Morphological Testing of the Thoracic Aortas with Electron Microscopy
The test was performed according to the procedure known from the literature (Br. J. of Pharmacol., 1995; 115, 415-420). 1 mm2 pieces of the aorta wall was cut out of the thoracic aorta of the rats, and were then fixed with 2.5% glutaraldehyde at room temperature for 2 hours. This was followed by a post-fixation with 1% osmium tetroxide for 1 hour. Afterwards, the tissue pieces were dehydrated with ethanol, and embedded in Durcupan ACM. The samples were evaluated qualitatively based on the images recorded on a Hitachi 7100 electron microscope. The results of the test are given in Table 3.
TABLE 3Electron microscopic examination of the compounds of theinvention on the thoracic aorta of SH rats (morphological testing)MaterialsDosesDegree of regenerationSH control, physiological saline solution1Compound no. 4., 20 mg/kg p.o.5Compound no. 8., 5 mg/kg p.o.5Compound no. 9., 5 mg/kg p.o.5Compound no. 11., 10 mg/kg p.o.4Compound no. 12., 20 mg/kg p.o.3Compound no. 13., 20 mg/kg p.o.4
The results of the morphological test are expressed on a scale of 1 to 5, depending upon the degree to which the treatment with various test compounds restored the hypertonia-induced endothelium damage, that is, upon the degree of regeneration activity observed. On the scale, 1 was used to refer to cases where no regeneration was observable, 2 refers to weak, 3 to average, 4 to good, and 5 to strong regeneration.
When comparing it to the untreated control, significant protective and regenerative effect was observed after treatment with the compounds of the invention. Due to the treatment, a thin, freshly formed layer covered the wounded sub-endothelium, which contained cells with active nuclei and rich cytoplasm. Regeneration was shown to be very effective in the case of the majority of the tested molecules.
Compounds of general formulae (I) where Y is a halogen atom are prepared by halogenating the suitable compound containing a hydroxyl group as Y substituent. The other compounds of the invention are prepared by the known method, according to the procedures given in WO 97/16439 and WO 98/06400. Methods for the preparation of certain compounds are demonstrated in the examples.
The compositions of the invention can be made in solid or liquid forms generally used in human and veterinary therapy. For oral administration tablets, coated tablets, dragé´es, granules, capsules, solutions or syrups, for rectal administration suppositories, and for parenteral administration lyophylised or not lyophylised injections or infusion solutions can be prepared by known preparation methods. The oral compositions may contain fillers such as microcrystalline cellulose, starch, lactose, lubricants, such as stearic acid and magnesium stearate, coating materials such as sugar, film materials such as hydroxymethyl cellulose, flavors or sweeteners such as methyl paraben or saccharine, and colorants. Auxiliaries in the suppositories may be for example cocoa butter and polyethylene glycol. The compositions for parenteral use may contain saline or optional dispersing and wetting agents such as propylene glycol along with the active ingredient.
The dose of the compounds of the invention depends on the illness of the patient and the disease and varies from 0.1 to 200 mg/kg/day, preferably from 0.1 to 50 mg/kg/day. For human therapy, the preferable oral dose is 10-200 mg, in case of rectal administration 1-15 mg, and in case of parenteral treatment 2-20 mg daily for adults. These doses are applied in unit dosage forms optionally distributed to 2-3 administrations, particularly in case of oral treatment.
The invention is demonstrated by the examples below.